Nefropati DM Patof 2011 Kanwar Et Al

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Annu Rev Pathol. 2011 ; 6: 395423. doi:10.1146/annurev.pathol.4.110807.092150.
A Glimpse of Various Pathogenetic Mechanisms of Diabetic

Nephropathy
Yashpal S. Kanwar1,2, Lin Sun2, Ping Xie1, Fu-you Liu3, and Sheldon Chen2
Yashpal S. Kanwar: y-kanwar@northwestern.edu; Lin Sun: sunlinkid@163.com
1Department of Pathology, Northwestern University School of Medicine, Chicago, Illinois 60611
2Department of Medicine, Northwestern University School of Medicine, Chicago, Illinois 60611
3Department of Nephrology, Central South University, Changsha, Hunan Province 410083, China
Abstract
Diabetic nephropathy is a well-known complication of diabetes and is a leading cause of chronic
renal failure in the Western world. It is characterized by the accumulation of extracellular matrix
in the glomerular and tubulointerstitial compartments and by the thickening and hyalinization of
intrarenal vasculature. The various cellular events and signaling pathways activated during
diabetic nephropathy may be similar in different cell types. Such cellular events include excessive
channeling of glucose intermediaries into various metabolic pathways with generation of advanced
glycation products, activation of protein kinase C, increased expression of transforming growth
factor and GTP-binding proteins, and generation of reactive oxygen species. In addition to these
metabolic and biochemical derangements, changes in the intraglomerular hemodynamics,
modulated in part by local activation of the renin-angiotensin system, compound the
hyperglycemia-induced injury. Events involving various intersecting pathways occur in most cell
types of the kidney.
Keywords
hyperglycemia; advanced glycation products; protein kinase C; GTP-binding proteins; reactive
oxygen species; hypertension; tubulointerstitial fibrosis
INTRODUCTION
Diabetic nephropathy is a known microvascular complication in patients with diabetes
mellitus, a metabolic disorder in which chronic hyperglycemia induces dysfunction in
various cell types of the kidney, ultimately leading to progressive renal failure (17). The
annual incidence of this disease has more than doubled in the past decade, and at present it
accounts for almost 50% of all end-stage renal diseases. Type 1 and type 2 diabetes are
distinct in pathogenesis, but the changes these disorders induce in various kidney
compartments namely the glomerulus, tubulointerstitium, and vasculatureare virtually
indistinguishable (7). Moreover, it seems that all the cell types of the kidney, including
glomerular podocytes, mesangial and endothelial cells, tubular epithelia, interstitial
fibroblasts, and vascular endothelia, are affected by hyperglycemic injury, although to
varying degrees. Because these cells are derived from ectodermal, endodermal, and
Copyright 2011 by Annual Reviews. All rights reserved

DISCLOSURE STATEMENT The authors are not aware of any affiliations, memberships, funding, or financial holdings that might
be perceived as affecting the objectivity of this review.
Kanwar et al. Page 2
mesodermal progenitors, this susceptibility may imply that hyperglycemia can induce injury
in cells of all three lineages; if so, the term microvascular complication may be a misnomer.
Certainly, the initial stages of diabetic nephropathy can be ascribed to dysfunctions of
glomerular capillaries, which clinically manifest as hyperfiltration and microalbuminuria (3,

8). Subsequent changes affect all the compartments and cell types of the kidney.
Well-described characteristic glomerular changes include mesangial cell proliferation and

hypertrophy, excessive accumulation of extracellular matrix (ECM) proteins in the form of
increased mesangial matrix, and thickening of the glomerular basement membrane (GBM),
which eventually leads to nodular glomeru-losclerosis, also known as Kimmelstiel-Wilson
lesions (Figure 1) (3, 9). Similar changes occur in the tubulointerstitial compartment,
including tubular hypertrophy followed by thickening of the tubular basement membrane
(TBM) and interstitial fibrosis (Figure 1d). The latter may be due to a unique process known
as epithelialto-mesenchymal transformation (EMT), which transforms tubular cells into
interstitial cells; this process causes excessive ECM to accumulate in the renal interstitium
(10, 11). Vascular changes typically include thickening and hyalinization of the afferent
arterioles and interlobular arteries with effacement of their endothelia.
Several lines of evidence suggest that all the cellular elements of the kidney respond to
hyperglycemic challenge by activating similar intracellular signaling events, albeit with
some variation depending on the expression of a given molecule in a particular cell type, for
instance, aldose reductase (AR) and myo-inositol oxygenase in the tubular cells and protein
kinase C (PKC)- and - isoforms in the glomeruli (1214). The intracellular events
induced in the presence of high-glucose ambience include accentuated flux of polyols and
hexosamines; generation of advanced glycation end products (AGEs) and reactive oxygen
species (ROS); activation of the PKC, transforming growth factor Smadmitogen-
activated protein kinase (TGF-SmadMAPK), and Janus kinasesignal transducer and
activator of transcription (JAK-STAT) pathways and of G protein signaling; aberrant
expression of cyclin kinases and their inhibitors; and aberrant expression of ECM proteins,
ECM-degrading enzymes, metalloproteinases, and their inhibitors (Figure 2) (9, 1524).
Cross talk among these events occurs at various levels in different signaling pathways. Some
of this cross talk, such as that between ROS and PKC, results in positive feedback signals
(25) that accentuate the hyperglycemia-induced injury and initiate certain deleterious
pathological processes, such as altered cell cycle and growth and excessive synthesis and
accumulation of ECM, a hallmark of diabetic nephropathy in humans (Figure 2). Another
important concept that is relevant to the pathogenesis of diabetic nephropathy is that
hyperglycemia impairs autoregulation within the glomerulus by activating the local
intrarenal renin-angiotensin aldosterone system (26, 27). This may increase the glomerular
capillary pressure and mechanical stretch on the mesangial cell and then activate signaling
molecules such as ROS, thereby amplifying various intracellular pathways and
synergistically worsening the progression of diabetic nephropathy. Such an outcome may be
regarded as a combination of metabolic and mechanical assaults on the kidneys within a
hyperglycemic milieu (Figure 2).
In this review, we discuss pathogenetic mechanisms relevant to hyperglycemia-induced

metabolic and mechanical renal injury. Overt diabetic nephropathy ensues following
insidious accumulation of various by-products generated during early acute metabolic
events, initiated by hyperglycemia, with disproportionate cellular intake of glucose.
CELLULAR UPTAKE OF GLUCOSE AND ITS PROCESSING INTO VARIOUS

METABOLIC PATHWAYS
Translocation of glucose into the renal cells is regulated by various facilitative transporters
such as glucose transporter (GLUT)-1 and 4, as well as by active energy-dependent
transporters, such as sodium-glucose-linked transporter 1 and 2; the latter transport glucose
via an electrochemical concentration gradient (28). The binding affinity/capacity kinetic
characteristics of GLUT-1 and 4 reveal that they can be readily saturated under normal
glucose concentrations, suggesting that their expression and/or activity may need to be
upregulated for high glucose concentrations to result in increased glucose transport across
the cell membrane; increased amounts of glucose would then be channeled into various
metabolic pathways, which would ultimately lead to increased expression of ECM proteins
such as type IV collagen and fibronectin. The hypothesis that increased expression of
GLUT-1 may contribute to the pathobiology of diabetic complications stems from studies of
GLUT-1-overexpressing mesangial cells; interestingly, these cells synthesize excessive
ECM even under normal glucose ambience (29). Conversely, downregulation of GLUT-1
with antisense treatment reduces glucose-induced fibronectin expression in mesangial cells,
which suggests that the very early steps of glucose transport can markedly influence
downstream cellular events and finally very distal processes, such as ECM synthesis (28).
Following the cellular uptake of glucose, this molecule undergoes phosphorylation through
conversion to glucose 6-phosphate (G6-P), which isomerizes to fructose 6-phosphate (F6-P);

then, after a series of reactions, glyceraldehyde 3-phosphate (G3-P) is formed (Figure 3)
(30). Through the actions of various transferases and phosphatases, G3-P forms glycerol
phosphate, a precursor of diacylglycerol (DAG), which by virtue of being a second
messenger is a well-known signaling molecule that is responsible for recruitment and
activation of PKC (31). Under high-glucose ambience, F6-P is diverted into the hexosamine
pathway, where it converts to glucosamine-6-phosphate by a rate-limiting enzyme,
glutamine:fructose-6-phosphate-aminotransferase (GFAT), through the synthesis of UDP-N-
acetylglucosamine, a precursor of ECM proteins such as the proteoglycans (32). The GFAT
also modulates promoter activities of ECM, which modulates TGF-1 and plasminogen
activator inhibitor 1 (PAI-1) by phosphorylating the RNA polymerasedependent
transcription factor Sp1 (32).
Excess glucose is also channeled into the accessory polyol pathway, where it is reduced to
polyalcohol sorbitol by AR, an NADPH-dependent enzyme (Figure 3) (16). The sorbitol is
oxidized to fructose by sorbitol dehydrogenase, which uses NAD+ as a cofactor. Under basal
conditions, minute amounts of glucose are handled via this route, but in a high-glucose
milieu, as much as 30% of the glucose is channeled through this pathway (16). The
accentuated activity of this pathway leads to relative depletion of NADPH and reduced
glutathione (GSH), an increase in the NADH/NAD+ ratio, and decreased levels of nitric
oxide (NO), which result in an altered cellular redox and therefore oxidant and osmotic
stress. The relevance of the polyol pathway in diabetic lesions is highlighted by studies in
which the eye lens of mice overexpressing AR exhibited reduced levels of GSH (16).
However, mice deficient in AR had normal GSH levels and nerve conduction velocity (33).
Also, the failure of AR inhibitors to ameliorate the cellular and matrix changes in the kidney
of diabetic rodents argues against an important and nonredundant role of the polyol pathway
in the pathogenesis of diabetic nephropathy (16).
Finally, glucose is channeled into another minor pathway, myo-inositol metabolism, that has
received little attention despite having been implicated in the pathogenesis of diabetic
nephropathy (34). L-myo-inositol,1-phosphate is generated from G6-P by the action of
enzyme synthase. Upon dephosphorylation, it forms myo-inositol, which is oxidized to D-
glucuronic acid by myo-inositol oxygenase (MIOX), which is expressed exclusively in the

kidney (13). In diabetic nephropathy, the tissue concentration of myo-inositol decreases as it
is increasingly excreted through the urine (13). At the same time, levels of Na+/K+-ATPase
also decrease, partly because of the increased flux of polyols and synthesis of this enzymes
inhibitors, namely arachidonic acid and prostaglandin E2, via PKC-induced activation of
phospholipase A2 (35). These biochemical abnormalities can be rectified by myo-inositol
supplementation (36). Likewise, addition of myo-inositol to cultures of proximal tubular
epithelial cells normalizes glucose-induced proliferation and collagen synthesis (37). Myo-
inositol deficiency reflects some combination of decreased synthesis and increased
degradation by the renal-specific enzyme MIOX; interestingly, MIOX is upregulated in
experimental diabetic nephropathy (13).
HYPERGLYCEMIA-INDUCED FORMATION OF ADVANCED GLYCATION

END PRODUCTS AND ENSUING RENAL INJURY
Initial steps in the synthesis of AGEs include nonenzymatic condensation of sugar and a free
amino group, which occurs through the formation of a labile Schiff base (Figure 4). The
Schiff base undergoes an intramolecular Amadori rearrangement followed by a series of
reactions and then dehydration and polymerization, which lead to the generation of
macromolecular forms of AGEs (38). AGEs are produced in small amounts under normal
physiological conditions, such as during aging, but their levels markedly increase in a
chronic hyperglycemic milieu in both the cellular and extracellular compartments in various
tissues, and more so in organs that are vascularized (39). Intracellularly, AGEs are derived
from various dicarbonyls, primarily methylglyoxal, which is synthesized from G3-P or
dihydroxyacetone following catalysis by G3-P dehydrogenase (GAPDH) (39). These AGEs
can modulate various intracellular events, such as the activation of PKC, MAPK and
transcription factors such as nuclear factor B (NF-B) (Figure 4) (40, 41). These events, in
turn, regulate the expression of diverse growth factors and cytokines such as TGF-, which
inevitably influence the synthesis of various ECM proteins.
Extracellular AGEs are formed by irreversible cross-linking of glucose with ECM structural
proteins, such as type IV collagen, laminin, fibronectin, and proteoglycans (41). These
modified proteins may have decreased susceptibility to enzymatic hydrolysis by matrix
metalloproteinases (MMPs), which would allow them to accumulate in the extracellular
space (24). Moreover, glycation of sulfated proteoglycans reduces their electronegativity
and thus modifies the charge-selective filtration properties of the basement membrane,
resulting in microalbuminuria (42, 43). In addition to adversely affecting ECM biology,
extracellular AGEs can modulate cellular functions by interacting with their cognate
receptor, RAGE, or with binding proteins, namely OST-48, 80KH, galectin-3, and type II
macrophage scavenger receptor, which may similarly alter cell and matrix functions (Figure
4) (39, 40). Such alterations may include interference with cell-matrix interactions as well as
changes in adhesiveness, neurite growth, and the hyperpermeability of capillaries (17).
Altered functions related to the vascular complications of diabetes mellitus can be partially
reversed (a) by the administration of aminoguanidine, an AGE inhibitor and AGE cross-link
breaker, or (b) by blocking RAGE, as has been highlighted in various experimental models
(41).
Finally, other altered cellular events relating to high concentrations of intra- or extracellular
AGEs in high-glucose ambience include the generation of ROS and quenching of NO; both
ROS and NO are likely to modulate PKC and MAPK activities and to activate transcription
factors such as NF-B and activator protein 1 (AP-1), thereby promoting a further increase
in the expression of ECM proteins (Figure 4) (3941). Intriguingly, AGEs themselves can
also covalently bind with ECM or cellular proteins, which further compound their
deleterious effects in various tissues (3941).
HYPERGLYCEMIA-INDUCED ACTIVATION OF PROTEIN KINASE C AND

RELEVANT CELLULAR SIGNALING
Among signaling kinases, PKC seems to be a centerpiece in the pathogenesis of diabetic
nephropathy (Figure 5) (14, 44). A number of PKC isoforms, including PKC-, -, -, -,
and -, are expressed in the kidney and are activated in diabetic rodents and in glomerular
cells studied in in vitro culture systems (45). PKC is activated by multiple routes, primarily
DAG, which is formed by the channeling of glycolytic intermediate dihydroxyacetone
phosphate to glycerol phosphate with subsequent removal of phosphate followed by
acylation (45). PKC activation also occurs during the increased activity of the polyol
pathway and following AGE:RAGE interaction (46). The latter interaction modulates the
activity of membrane-bound phospholipase C, leading to an increase in cytosolic Ca2+ and
DAG; these molecules may then act as second messengers, which could further boost PKC
cellular signaling. This mechanism would imply a close relationship between the cellular
levels of DAG and the extent of PKC activation. Indeed, such a correlation has been
observed in various tissues, including renal mesangial cells subjected to high-glucose
ambience (47).
PKC activation can influence a number of downstream pathophysiological changes, such as

altered blood flow and capillary permeability, which may be due to (a) decreased expression
of endothelial nitric oxide synthase (eNOS) and NO and (b) upregulation of endothelin 1
and vascular endothelial growth factor (VEGF), as exemplified by studies of eNOS
knockout mice (Figure 5) (48). Vascular compromise may be accentuated by greater buildup
of ECM proteins due to increased PKC-induced TGF-1 expression and activation of
plasmalemmal NADPH oxido-reductase; the latter may thereby induce cellular oxidant
stress (18). The vascular injury may be further compounded by PKC-induced
overexpression of PAI-1, which is an inhibitor of fibrinolysis, and concomitant activation of
NF-B, resulting in a severe inflammatory response and thrombotic angiopathy (25, 49).
These cellular events suggest that PKC plays a pivotal role in the pathogenesis of
microvascular complications of diabetes. That the administration of ruboxistaurin mesylate,
a PKC inhibitor with affinity for the 1 and 2 isoforms, partially reduces renal damage in
db/db mice (a model of type 2 diabetes) supports a central role for PKC in hyperglycemia-
induced renovascular injury (50).
REACTIVE OXYGEN SPECIES MODULATION OF OTHER

HYPERGLYCEMIA-INDUCED CELLULAR SIGNALING EVENTS

Recent studies suggest that the activities of AGEs, PKC, and ROS are interrelated and that
the latter may serve as reciprocal inducers and amplifiers of the signaling cellular events that
occur in high-glucose ambience (25, 42, 49, 51, 52). Normally, ROS are produced in minute
amounts that are necessary to maintain cellular homeostasis, but in states of hyperglycemia
their concentrations rise dramatically, damaging various target organs (49). The ROS that
can induce renal injury include superoxide anion O2, H2O2, hydroxyl radical, and
peroxynitrite (52). Enzymes that can scavenge ROS include cytoplasmic CuZn superoxide
dismutase (CuZnSOD), mitochondrial manganese superoxide dismutase (MnSOD), and
heme oxygenase 1 (53, 54). Interestingly, the latter undergoes a remarkable adaptive
response (> 15-fold increase) in states of hyperglycemia, presumably to reduce ROS-
mediated oxidant stress (54). ROS are generated via two systems: (a) predominantly via
mitochondrial oxidative phosphorylation and (b) in small amounts via the NADPH-oxidase
system (5558).
ROS are generated as a by-product of oxidative phosphorylation, in which electron donors

such as NADH and FADH2 generate a high membrane potential by pumping protons across
the mitochondrial inner membrane (Figure 6) (42, 59). As a result, electron transport is
inhibited, the half-life of free-radical intermediates of coenzyme Q increases, and molecular
O2 is reduced to O2 with ensuing oxidant stress. In support of this hypothesis are results
from studies in which (a) rotenone, an inhibitor of electron transfer chain complex I, and (b)
m-cholorophenyl-hydrazone, an uncoupler of electron transport, blocked the generation of
dichlorofluorescein-sensitive ROS (42). Similar results were observed following the
overexpression of mitochondrial MnSOD or uncoupling protein 1 (42, 60). MnSOD or
uncoupling protein 1 may reversibly modulate the four major cellular events induced by
high glucose that can induce oxidant stress: polyol and hexosamine flux, AGE formation,
and DAG-induced PKC activation (Figure 6). The O2 that is generated initially inhibits
GAPDH, which in turn amplifies the above-described critical cellular events through the
cyclic generation of ROS (25). That gene disruption of GADPH also amplifies the above-
named cellular events lends support to the hypothesis that GADPH plays a critical role in the
generation of mitochondrial ROS (61).
The relevance of extramitochondrial ROS to the pathogenesis of diabetic nephropathy is

supported by studies in which overexpression of cytoplasmic CuZnSOD reduced glomerular

pathophysiologic changes in db/db mice and in mice with streptozotocin (STZ)-induced
diabetes (62, 63). Extramitochondrial ROS are generated via the NADPH-oxidase system,
which is normally dormant but that, upon association of membrane-bound flavocytochrome
b558 (heterodimer of gp91phox and p22phox) with various cytosolic proteins (p47phox;
p67phos; p40phox; and a GTP-binding protein, p21rac), generates O2 and H2O2 (51, 56, 57).
Among these cytosolic proteins, Nox4 is particularly interesting in renal pathobiology
because it is a homolog of neutrophil gp91phox, which is expressed in the kidney (64).
Similar to the mitochondrial electrontransport system, the NADPH-oxidase system can be

activated in renal cells by AGEs, PKC, DAG, and IP3 (inositol 1,4,5-triphosphate) in renal
cells. In addition, the metabolites of the cyclo-oxygenase pathway can activate NADPH
oxidase under high-glucose ambience (49, 65). The fact that fibronectin expression
decreases in tubular or mesangial cells treated with inhibitors of oxidase, namely apocynin
and diphenylene iodonium, suggests that NADPH oxidase may play a role in high glucose
mediated injury and supports its relevance in redox-sensitive processes, such as cell growth,
apoptosis, migration, and ECM modeling (66, 68). ECM modeling can be markedly
modulated by diverse signaling pathways, transcription factors, and growth factors (6971).
TGF-, a potent cytokine, may adversely affect the biology of tubular cells via the induction
of EMT and loss of epithelial cell adhesion, smooth muscle actin expression, cytoskeletal
organization, and cell migration across the basement membrane, as well as by promoting
tubulointerstitial fibrosis, which is an integral part of diabetic nephropathy (72, 73).
Finally, special forms of ROS, known as reactive nitrogen species (RNS), are produced
following hyperglycemia-induced endothelial injury, in which a series of events leads to the
disruption of the nitroso-redox balance (74). NO synthesis is modulated by a cofactor of
eNOS known as tetrahydrobiopterin (BH4) (75, 76). Under high-glucose ambience, BH4
levels are reduced, with a proportionate decrease in the synthesis of NO by the endothelium
and an altered ratio of BH4 to its oxidized form, BH2. These reductions eventually lead to
increased generation of O2 (77). That exposure of diabetic rat aortic-ring explants to BH4
ameliorates endothelialsmooth muscle dysfunction supports a role for RNS in the
pathogenesis of hyperglycemic injury (77).
TRANSFORMING GROWTH FACTOR CELLULAR SIGNALING IS CRITICAL

TO THE INDUCTION OF GLOMERULAR AND TUBULOINTERSTITIAL
FIBROSIS IN DIABETIC NEPHROPATHY

TGF-1 is widely thought to be the most pertinent cytokine to the ECM glomerular
pathology typically observed in patients with chronic progressive diabetic nephropathy.
However, most of the consequences of TGF-1-related signaling (Figure 7) have been
identified in studies of cultured glomerular mesangial cells and podocytes in vitro (19, 23,
78, 79). TGF-1, a prototype of the TGF- superfamily, exerts pleiotropic effectsthat is, it
inhibits proliferation in certain cells and apoptosis in othersbut it induces hyperplasia and
hypertrophy of mesangial cells (19, 23, 78, 79). In the ECM, TGF-1 exists as a latent,
dormant form of propeptide complexed with TGF-1-binding proteins, and both are cross-
linked with matrix proteins by transglutaminase (6871). Following cleavage by plasmin,
MMP-2 and 9 or thrombospondin 1, an active free form of TGF-, are generated.
The active TGF-1 binds first to a type II serine/threonine kinase receptor, which
transphosphorylates and thereby activates a type I receptor. This process is followed by
modulation of the downstream-signaling Smad, MAPK, and perhaps protein kinase A
cellular pathways and various nuclear events (Figure 7). The activated TGF-1 receptor
interacts with Smad2 and 3 to form a heterodimeric complex with common partner co-
Smad4, which translocates into the nucleus and regulates transcription of TGF-1 target
genes such as collagen 1(I), PAI-1,Jun B, c-Jun, and fibronectin (6871). TGF-1-induced
Smad2/3/4 signaling has a counterregulatory loop driven by Smad7; the latter is modulated
by NF-B or Jak1/Stat1, which are activated by inflammatory cytokines (20, 21, 67).
Along with the Smads, the extracellular signal-regulated kinases 1 and 2 (ERK1 and 2), the
p44/p42 MAPKs, c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK), and
p38 MAPK modulate the TGF-1 signaling cascade in mesangial cells (19). These kinases
also regulate the transcriptional regulation of pro1(I) procollagen and fibronectin via AP-1,
a heterodimer of the c-Fos and c-Jun family members (80). AP-1, regulated by ERK and
JNK, can complex with Smad3, whereas the Smad3/4 complex can bind to AP-1 consensus
sequences in various promoters of TGF- target genes. These findings suggest that cross
talk occurs between the Smad and MAPK pathways and that TGF- signaling is central to
ECM pathobiology in diabetic nephropathy (19, 81).
TGF-1 signaling is initiated by numerous mediators generated under high-glucose

ambience, such as the AGEs, ROS, DAG, PKC, and the hexosamines; other mediators that
accelerate renal injury include vasoactive substances, such as angiotensin II, endothelin, and
thromboxane, as well as the physical cyclical stretching and relaxation of mesangial cells
(which mimic intraglomerular hypertension) (31, 82). The net effect is an increased
synthesis of various matrix proteins and accumulation of ECM. The amassing of ECM may
also be related to the inhibition of MMPs and the activation of tissue inhibitors of MMPs
(TIMPs).
The expression of ECM genes is also modulated by another cytokine, connective tissue
growth factor (CTGF), which is induced by TGF-1 via consensus sequences of Smad and
transcription enhancer factor elements that are present in the CTGF promoter (83, 84).
CTGF promotes ECM production, cell adhesion, and collagen matrix contraction in various
mesenchymal cells, such as cultured dermal fibroblasts and mesangial cells (83, 85). The in
vitro effects of TGF-1 have been well corroborated by in vivo studies, in which
upregulation of TGF- messenger RNA (mRNA) and protein expression, as well as its type
II receptor and TGF-1 bioactivity, were observed in the kidneys of various murine models
of diabetes (86, 87). That administration of neutralizing anti-TGF-1 antibodies prevents

renal hypertrophy, mesangial matrix expansion, increase in 1(IV) collagen and fibronectin
expression, and decline in renal function in mice with STZ-induced diabetes suggests a
potential role for TGF-1 in the pathogenesis of diabetic nephropathy (88). Similar
observations have been made in db/db mice with genetic type 2 diabetes mellitus (89).
Moreover, upregulated renal expression and increased urinary excretion of TGF-1 have
been reported in patients with diabetic nephropathy (90). Interestingly, angiotensin-
converting enzyme (ACE) inhibitors that protect the kidney from end organ damage also
reduce TGF-1 production, thus leaving little doubt that this profi-brogenic cytokine is
intricately involved in the renal pathobiology of diabetes (91).
Bone morphogenetic protein 7 (BMP-7), a member of the TGF- superfamily, acts in

opposition to the classical profibrogenic effects of TGF-1 (92), perhaps because some of
the BMPs induce Id proteins that inhibit differentiation or DNA binding via the Smad1/5
pathway, and these proteins may act as negative regulators of various basic helix-loop-helix
transcription factors (93). BMP-7 is expressed early in embryonic life, when it plays a
crucial role in eye and kidney development (92). In adult life, it is highly expressed in the
tubular epithelia of the outer cortex and in glomerular podocytes (94, 95). The protective
antifibrogenic role of BMP-7 in diabetic nephropathy has been demonstrated in transgenic
mice, where its expression was driven by the phosphoenolpyruvate carboxykinase
promoter (96). These mice had reduced glomerulosclerosis and tubulointerstitial fibrosis
when made diabetic by the administration of STZ. In addition to having reduced renal
expression of collagen and fibronectin, the mice demonstrated reduced dropout of
podocytes, reduced urinary protein (associated with partial restoration of nephrin
expression), and decreased serum creatinine levels; these results suggested that there may be
an imbalance in the activity among various members of the TGF- superfamily in diabetic
nephropathy.
The balance between ECM synthesis and degradation is also maintained by MMPs and
TIMPs. Interestingly, glomerular mRNA levels of MMP-2 and MMP-3 are decreased in rats
with STZ-induced diabetes, whereas TIMP mRNA levels are increased (24). These findings
suggest that profibrogenic cytokines, such as TGF-1, and MMPs and TIMPs play
intertwined and complex roles in the pathogenesis of ECM accumulation during diabetes.
EMERGING RELEVANCE OF GTP-BINDING PROTEINS AND CELL-CYCLE

PROTEINS IN VARIOUS PATHOGENETIC MECHANISMS OF DIABETIC
NEPHROPATHY
To date, few reports in the literature have described the role of GTP-binding proteins in the
pathogenesis of diabetic nephropathy. The major GTPases studied so far are the Ras, Ras-
related, and Rho families of GTP-binding monomeric proteins, which range from 20 to 40
kDa in size and belong to a superfamily that comprises more than 100 small GTPases (97).
They modulate a wide variety of cellular processes, including cell hypertrophy,
morphogenesis, motility, axonal guidance, cytokinesis, and intracellular trafficking (51, 97,
98). By serving as molecular switches in these processes, they cycle between inactive (GDP-
bound) and active (GTP-bound) states (97, 98). The state of activity is determined by the
action of guanine exchange factors, whereas GTPase-activating proteins (GAPs) facilitate
hydrolysis to GDP with a resulting loss of activity (Figure 8).
Among members of the GTPase superfamily, the Ras, Ras-proximate 1 (Rap1), and Rho
families are of particular interest because they are pivotal to many transduction pathways.
For instance, in the Ras/Raf/MEK (MAPK/ERK) signaling pathway, Ras serves as an
intermediary between the activated/phosphorylated growth factor receptor and MEK,

whereas the JNK/SAPK and P38/MAPK pathways may be directly activated by Rho and
Rho-related Rac, respectively (22, 51, 99). Interestingly, Rap1 and Ras are in
counterequilibrium, and their activity state depends on association with another serine/
threonine kinase, Raf, which is activated by the ligand-induced phosphorylated form of the
growth factor receptor. For instance, following insulin stimulation, Rap1 is inactivated by its
dissociation from Raf, and the free Raf associates with GTP-bound activated Ras (100).
However, insulin deficiency leads to activation of Rap1 through its association with Raf.
Rap1 can also be activated by second messengers such as Ca2+, cAMP, and DAG.
Activation of the Rap1/Raf/MAPK pathway can also occur through PKC and ROS that are
generated following AGE:RAGE interaction (97, 98). Such signaling mechanisms have been
elucidated both in vitro and in vivo through the upregulation of Rap1b in high-glucose
ambience, which led to increased ECM-fibronectin synthesis (Figure 8) (101, 102).
Posttranslational lipid modifications of Rap1b, such as farnesylation, may also be critical to
Rap1 activation and translocation to the inner plasmalemmal leaflet in that they may
ultimately have an effect on ECM protein synthesis. Similar modifications also occur in the
Rho GTPases, but the latter undergo geranylgeranylation rather than farnesylation (100,
103).
The Rho family of proteins regulate cytoskeletal organization, actin polymerization, and cell
migration and adhesion (51, 97, 98). However, recent findings suggest that the Rho GTPases
play a pivotal role in the pathobiology of ECM proteins, such as fibronectin, that are
modulated by TGF--induced upregulation of CTGF, a powerful profibrogenic cytokine
expressed in renal glomerular and tubulointerstitial cells (100, 104). Similarly, Rho-
dependent pathways are activated in the kidney by other profibrogenic molecules, including
angiotensin II, platelet-derived growth factor, and endothelin 1 (Figure 8) (100, 105). These
factors modulate the expression of various ECM proteins and thus are implicated in the
pathogenesis of diabetic nephropathy. The relevance of Rho GTPases to diabetic
nephropathy is further underscored by the fact that Rho GTPases activate JNK/SAPK, which
can influence the activity of transcription factors such as Elk1, nuclear transcription factor,
AP-1, NF-B, Myc, and cAMP-responsive elementbinding protein (CREB) (106, 107).
The cis-acting cAMP-responsive elements, TGACGTCA, have been localized in the
promoters of various ECM proteins, and therefore CREB could be regarded as the major
transcription factor responsible for their increased expression in the diabetic state (Figure 8)
(108).
GTPases have also been implicated in the modulation of other cellular processes, such as
apoptosis, differentiation, and cellular proliferation, through the activation of JNK/SAPK
and P38/MAPK and stimulation of serum response factordependent transcription, which is
regulated by generic transcription factors such as AP-1 and NF-B (51, 109). Such
processes, especially apoptosis in tubular cells and the proliferation of mesangial cells, have
been described as early features of diabetic nephropathy and evidently are modulated by
various cell-cycle proteins and their inhibitors (23, 99, 103). Cell-cycle proteins are a
complex of cyclins and cyclin-dependent kinases (CDKs); the latter are activated upon
heterodimerization with cyclins (110, 111). Levels of these cyclins fluctuate during the cell
cycle, and their expression may be regulated by growth factors, whereas CDKs are
constitutively expressed, and their activity depends upon their state of phosphorylation and
their binding to cyclins (112). The activity of the cyclin/CDK heterodimer complex is
negatively regulated by another group of small proteins, known as CDK inhibitors (CDIs).
CDIs comprise two main families, namely Cip/Kip (p21Cip1and p27Kip1) and 1NK4/ARF
(p161NK4 and p14ARF) (110, 111). The role of cell-cycle proteins in diabetic nephropathy
was first demonstrated in studies in db/db mice, a model of type 2 diabetes, in which
investigators observed an increased expression of p27Kip1 relative to that of heterozygous
db+ littermates (111). Likewise, kidneys of mice with an STZ-induced type 1 model of
diabetes demonstrated a similar increased expression of CDIs (113). Mesangial cells
exposed to high-glucose ambience also showed increased expression of p27Kip1, which may
have been related to this proteins phosphorylation by MAPKor PKC, both of which are
activated in a diabetic milieu (111). Interestingly, exposure of mesangial cells to a gene-
specific phosphorothioated antisense oligonucleotide resulted in decreased protein synthesis
of p27Kip1, suggesting that a phenotypic conversion from the hypertrophic phase to a
hyperplastic state takes place for cells exiting the G1 phase and entering the S phase of the
cell cycle (113). Such a phenotypic change has also been reported in in vivo studies, in
which induction of diabetes in p27Kip1/ or p21Cip1/ mice resulted in a hyperplastic rather
than hypertrophic response in tubular cells (111, 114). The inability of STZ to induce overt
renal lesions in p27Kip1/ mice also underscores the significance of cell-cycle proteins in
the pathogenesis of diabetic nephropathy (114). Further support for the role of cyclins in this
setting is provided by studies in which decreased expression of p21Cip1 was associated with
a reduction in kinase activities of CDK-2 and 4 in mesangial cells undergoing proliferation
under high-glucose ambience (103). In such experiments, there was a concomitant increased
expression of Ras and Rho, which were subsequently translocated to the inner leaflet of the
plasma membrane. Such findings support the importance of cell-cycle proteins and the
GTPase/p21 signaling pathway in the cellular response to hyperglycemic stress (103, 105).
ROLE OF INTRAGLOMERULAR HYPERTENSION IN THE AMPLIFICATION

OF HYPERGLYCEMIA-INDUCED RENAL INJURY IN DIABETIC

NEPHROPATHY
A well-recognized early pathophysiologic feature of diabetic nephropathy is hyperfiltration,
which is reflected in a marked increase in the glomerular filtration rate originally described
by Anderson & Brenner (115). This increase seems to result from intrarenal hypertension or,
more specifically, from a rise in glomerular capillary pressure. The increased pressure may
accelerate the renovascular complications, perhaps via cross talk between metabolic and
hemodynamic factors in a diabetic milieu. This important hypothesis, which has evolved in
recent years (116), arose from seminal micropuncture studies in which rats with
experimental diabetes had intraglomerular hypertension despite having normal systemic
blood pressure; this finding indicated that the autoregulation of local glomerular
microcirculation was impaired because of the differential dilatation of arterioles (afferent
versus efferent), which led to an increase in the transcapillary hydraulic pressure difference
and plasma flow (117). Hyperglycemia may expose the kidney to a local increase in blood
pressure, probably by activating the reninangiotensin system (RAS) with regional renal
production of angiotensin II (82). That significant amelioration in the microalbuminuric
response occurs upon reduction of the intraglomerular hypertension in states of

hyperglycemia supports the above hypothesis (118).
Intriguingly, the intrinsic cells of the glomerulus, such as mesangial cells and podocytes,
synthesize angiotensin II and express angiotensin II receptors, which may contribute to the
regional activation of RAS, leading to mechanical pressureinduced damage and thereby an
accentuation and perpetuation of hyperglycemia-mediated ROS or glycative stress injury to
the kidney (Figure 9) (119123). These proposed mechanisms favor the inhibition of ACE
as an integral part of the therapy for amelioration of diabetic nephropathy. Lending support
to this idea are ACE gene insertion/deletion polymorphism studies, which indicate that
individuals with the ACE deletion/ deletion genotype respond poorly to ACE inhibition; this
possibility suggests that in the setting of metabolically induced altered hemody-namics,
genetics may be critical to determining the outcome of this disease (124). The relevance of
hemodynamics is further suggested by animal studies in which a deficiency of ACE2, a
negative regulator of ACE, is associated with a local increase of tubular angiotensin II and
increased tubulointerstitial fibrosis in long-term experimental diabetes (125). That ACE2
knockout mice tend to develop glomerulosclerosis that is reminiscent of diabetic
nephropathy also underscores the importance of ACE2 and intraglomerular hemodynamics

in the pathogenesis of diabetic nephropathy (126).
As noted above, there are a multitude of pathways involved in the pathogenesis of diabetic
nephropathy. However, a critical issue is identifying the mechanism(s) by which
hyperglycemia induces the kidney to generate increased amounts of angiotensin II that
exacerbate the hemodynamic injury. Increased production of angiotensin II is thought to be
central to the early hyperplasia and late hypertrophy of the renal cells observed in diabetes,
which occur through the upregulation of cytokines such as TGF-, CTGF, interleukin-6,
monocyte chemoattractant protein 1 (MCP-1), and VEGF-A, which modulate glomerular
ECM pathobiology (Figure 9) (3, 82, 117). First, with respect to angiotensin II synthesis,
high glucose upregulates the expression of renin and angiotensinogen in mesangial cells,
which could increase intrarenal concentrations of angiotensin II and then, via various
autocrine and paracrine pathways, lead to the generation of various cytokines and to ECM
accumulation (127). Second, the ROS generated in various hyperglycemia-mediated cellular
events, such as AGE:RAGE interactions, could also increase the expression of
angiotensinogen and angiotensin II in renal cells (17, 128). Infusion of AGEs in vivo causes
a remarkable increase in the expression of various components of the RAS (128). However,
infusion of angiotensin II increases both serum and renal concentration of AGEs, thereby
highlighting the complexity of the cellular events that occur in a high-glucose environment
(128).
The angiotensin IIAGE juxtacrine loop may maintain a relatively constant increase in the
glomerular capillary pressure by affecting the efferent arteriole, thereby transmitting a
sustained-stretch stress on the glomerular cells, which may lead to parallel changes in the
glomerular volume and to the activation of various signaling pathways and a rise in blood
pressure (Figure 10) (124126). Such an unrelenting mechanical stretch would adversely
affect the biology of mesangial cells in several ways: First, it would induce the expression of
GLUT-1 through an increase in cellular glucose concentration, despite a normal
extracellular glucose ambience in the culture medium (123, 129). Second, recurring cyclic
stretch and relaxation of mesangial cells in vitro enhance these cells proliferation and
increase the synthesis of ECM while decreasing the expression of ECM-degrading enzymes
(130). Third, the mechanical stretch could either directly induce the expression of TGF-
and its receptor or indirectly activate PKC and p38/MAPK with consequential increased
synthesis of various ECM proteins (131). Another significant angiotensin IImediated
stretch stress response in mesangial cells includes the induction of MCP-1 (132), which
upregulates intercellular adhesion molecule 1 and the generation of ROS, which may lead to
amplification of the inflammatory response and renal injury. That the influx of monocytes,
and their adherence to the glomerular mesangium, is often observed in clinical renal biopsies
accords with the notion that inflammatory cytokines also play a role in the pathogenesis of
diabetic nephropathy (133). In addition to mesangial cells, podocytes are adversely affected
by the sustained glomerular capillary stretch, possibly via the induction of VEGF-A (79,
134). Such stress may lead to a reversible reorganization of the cytoskeletal assembly and to
reduced expression of 31 integrin in podocytes. As a consequence, podocytes may detach
from the underlying GBM and undergo apoptosis, leading to ensuing proteinuria (79, 135).
VEGF signaling reduces the expression of an important glomerular slit-membrane protein
known as nephrin (135, 136). Nephrin regulates the transcapillary passage of plasma
proteins, and its loss may accentuate the proteinuric response during diabetes (137).
Interestingly, decreased expression of nephrin with relative loss of podocytes has been
observed in neonates of diabetic mothers, and such underdosing of nephrons with nephrin
and the attendant podocytopenia during fetal development may predispose patients to
developing hypertension later in adult life (138). Key metabolic and hemodynamic events
are set in motion by hyperglycemia, and in the setting of a sufficient genetic predisposition,
such events could cyclically amplify to give rise to the renovascular complications of
diabetic kidney disease.
PATHOGENETIC MECHANISMS RELEVANT TO TUBULOINTERSTITIAL

INJURY IN DIABETIC NEPHROPATHY
Until recently, studies to delineate the pathogenesis of diabetic nephropathy have focused on
the renal glomerulus, and only a few reports have addressed the pathobiology of the
tubulointerstitium (10, 73, 139142). Although the tubulointerstitium is thought to be
adversely affected at a later stage during the evolution of diabetic nephropathy than the
glomerulus, the extent of the damage to this compartment correlates much better with renal
dysfunction (e.g., decrease in the glomerular filtration rate) than does damage to the
glomerular compartment (143). That the tubulointerstitial compartment represents the vast
majority (90%) of the parenchymal mass of the kidney reinforces the importance of this
compartment to the pathogenesis of diabetic nephropathy. For example, in the initial stages
of diabetic nephropathy, when hyperfiltration and glomerular enlargement are prevalent, the
renomegaly observed is related to the notable hypertrophy of the tubules (115, 142).
Similarly, damage to this compartment in the later stages of diabetic nephropathy, albeit by
varied pathogenetic mechanisms, may substantially impair renal function (142, 143).
Some mechanisms relevant to tubulointerstitial pathobiology may be similar to those

described above for the renal glomerulus, but certain processes can selectively affect this
compartment. The pathogenetic mechanisms that are common to both glomerular and
tubulointerstitial compartments include increased channeling of glucose into the polyol
pathway; generation of AGEs and ROS;and activation of various pathways pertaining to or
influencing RAS, PKC-, and TGF- signaling. The actions of these mechanisms are
supported by both in vivo and in vitro studies. For instance, direct exposure of tubular HK-2
cells to high-glucose ambience increases TGF- activity and collagen production (37, 144).
Also, in situ hybridization and immunohistochemical studies indicate that, with the
inhibition of ACE or PKC-, there is a significant decrease in TGF- activity and in the
expression of collagen and osteopontin, which is another ECM protein confined mainly to
the tubulointerstitium (145, 146).
Tubulointerstitial injury may also occur secondary to adverse changes in the glomerulus,
such as proteinuria observed in later stages of diabetic nephropathy (10, 140). Proteinuria,
especially the nonselective type, can induce tubulointerstitial damage by several different
mechanisms (147). First, a glomerular filtrate containing excessive amounts of profibrogenic

cytokines would adversely affect the biology of tubular epithelium and interstitial cells (140,
148). Second, protein reabsorption overload in the tubular epithelium may cause lysosomal
rupture, increased energy demand, and ultimately apoptosis. Third, tubular epithelial cell
injury may be further augmented by increased concentrations of various activated
components of the complement system and its endogenous regulatory proteins in tubular
lumina, such as C3, C3a, C5a, C5b9, and Crry (10, 149). Several of these components have
chemotactic properties and therefore may generate an inflammatory response that leads to
elaboration of MCP-1 and induction of an influx of monocytes, which consequently damage
the tubulointerstitium. Support for this hypothesis comes from studies in which blockade of
complement or MCP-1 activity ameliorated tubulointerstitial injury (150). Fourth, as in other
chronic kidney diseases, proteinuria may be associated with transdifferentiation of tubular
cells into myofibroblastsa phenotypic change that is described in the literature as EMT
(10, 151153). Interestingly, the EMT is mediated by integrin-linked kinase and is
stimulated by CTGF. The latter promotes fibrosis and sclerosis in the glomerular
compartment, and its activity is modulated by TGF- (8385, 148, 154).
Another recently identified mechanism by which tubulointerstitial damage can occur in

diabetic nephropathy relates to hypoxia-induced injury, irrespective of the presence or
absence of proteinuria. Usually, hypoxia is attributed to chronic ischemia, which may occur
by (a) intrarenal vasoconstriction secondary to local activation of RAS or loss of NO or (b)
structural impairment of blood flow (10, 142, 155). The latter may occur secondary to
interstitial fibrosis, which encases the peritubular capillaries in scarred tissue and thus
restricts delivery of oxygen to the tubules (10, 156). Similarly, increased oxidant stress,
along with a deficiency of NO, would further exacerbate capillary endothelial dysfunction,
resulting in hypoxia of the tubulointerstitial compartment (157, 158).
Hypoxia is likely to induce functional impairment in the mitochondria of the tubular cells;
the ensuing apoptosis is frequently observed in animal models of diabetic nephropathy as
well as in humans (159). Also, hypoxia itself may induce activation of resident interstitial
cells and EMT of the tubular cells that accelerate fibrosis with further compromise to the
peritubular oxygen delivery (10, 156). In support of this idea, in vitro studies of renal
interstitial fibroblasts subjected to oxygen deprivation demonstrated an increase in the
transcription of collagen genes and TIMP-1 (160). The increase in TIMP-1 expression was
related to the increased activity of its promoter, which is regulated by a transcription factor
known as hypoxia-inducible factor 1 (HIF-1) (160, 161). Interestingly, HIF-1 can also bind
to the promoter region of the profibrogenic cytokine CTGF, which could promote fibrosis
(161). HIF-1 is a versatile transcription factor that modulates a number of pathways and the
expression of several genes, includingVEGF and erythropoietin (EPO) (162165). In the
initial stages of hypoxia, the intact peritubular cells may produce sufficient EPO to maintain
hemoglobin levels and O2 tension in the interstitium. However, with sustained damage by
chronic hypoxia, hyperglycemia, oxidant stress, and endothelial dysfunction, loss of the
EPO-producing fibroblasts may occur, along with anemia and progression of interstitial
fibrosis, thereby initiating a vicious cycle of hypoxia and tubulointerstitial injury (10, 142,
156).
SUMMARY
Diabetic kidney injury begins with hyperglycemia, the sine qua non of diabetes mellitus.
What happens as a result of hyperglycemia is a complex perturbation of cellular functioning
and extracellular occurrences and hemodynamic effects that collectively alter the biology of
virtually every type of kidney cell, whether in the vasculature, the nephron proper, or the
surrounding interstitial parenchyma. The unassuming glucose molecule, when processed in
greater flux through its diverse metabolic pathways, gives rise to DAG, GFAT,
hexosamines, polyols, an altered redox environment, Amadori adducts, AGEs, and ROS.
These molecules in turn trigger a cascade of signaling events that end up being maladaptive.
Specifically, PKC, ERK, p38, JNK/SAPK, the small GTPases, AGE:RAGE signaling,
angiotensin II, MCP-1, VEGF-A, and TGF-1 are intertwined in a web of cross talk,
impingements, activations, and repressions. As a result, the balance of cyclin kinases and
transcription factors, such as AP-1, NF-B, and Smads, tips in favor of a metabolic program
that causes cellular hypertrophy, generates ECM, favors phenotypic switching, and
selectively induces apoptosis. These processes give rise to the diabetic nephropathic
manifestations of renomegaly, mesangial matrix expansion, Kimmelstiel-Wilson lesions,
GBM thickening, podocytopenia, TBM thickening, interstitial fibrosis, and arteriolar
hyalinization (Figure 1). However, these conditions represent only a fraction of the
complexities of the renal cellular machinery. A complete description of the pathogenesis of
diabetic nephropathy does not end here. Research challenges for the future may include the
forging of new links between the metabolic and hemodynamic events and the elucidation of
how all these myriad events interact to produce the clinical features of hypertension,
proteinuria, and chronic kidney failure. Ongoing studies are beginning to elucidate the role
of the GTP-binding proteins and are emphasizing the relevance of the tubulointerstitium,
along with that of the mitochondrial-emanated oxidant stress, in diabetic nephropathy (166).
Finally, other unresolved issues that are currently being investigated in many research
laboratories and that are also important topics for future discussion include the role of
macrophage, epigenetics, and microRNA in the pathogenesis of diabetic nephropathy (167
169).
Acknowledgments
The authors work is supported by National Institutes of Health grants DK28492, DK44513, and DK60635.
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Figure 1.
Light photomicrographs illustrating various stages of developing glomerular lesions and
tubulointerstitial disease in diabetic nephropathy. (a) A normal glomerulus. (b) Thickened

basement membranes (arrowheads) and expanded mesangial regions (asterisks). (c) The
nodular appearance of the mesangial regions characteristic of Kimmelstiel-Wilson lesions
(asterisks). (d) The tubulointerstitial lesions include thickened tubular basement membrane
(TBM), hyalinization of afferent arteriole (ART), and fibrosis of the interstitium (INT).
Abbreviations: c, capillary lumen; En, endothelial cell; Ep, visceral epithelial cell
(podocyte); Me, mesangium; US, urinary space.
Figure 2.
An overview of different signaling events induced by exposure of renal cells to high glucose
concentrations, with resulting altered expression of various genes and cellular abnormalities
leading to diabetic nephropathy. The schematic drawing also highlights the hypothetical
cross talk between AGE-RAGE (advanced glycation end productreceptor for AGE) and the
renin-angiotensin system (RAS) and the reciprocal-cyclical modulation of the interactions
among AGEs, reactive oxygen species (ROS), and protein kinase C (PKC), with ROS as the
central mediator. Abbreviations: Ang II, angiotensin II; AP-1, activator protein 1; AT1, Ang
II receptor; ECM, extracellular matrix; GLUT, glucose transporter; JAK-STAT, Janus
kinasesignal transducer and activator of transcription; MAPK, mitogen-activated protein
kinase; MCP-1, monocyte chemoattractant protein 1; NF-B; nuclear factor B; RNS,

reactive nitrogen species; TGF-, transforming growth factor ; VEGF, vascular endothelial
growth factor.
Figure 3.
Schematics of different pathways for glucose metabolism that lead to the activation of
protein kinase C (PKC) and transforming growth factor (TGF-). In addition to undergoing
glycolysis, excess glucose is channeled into the polyol and hexosamine pathways, which
results in increased lipid synthesis and the generation of diacylglycerol (DAG). This leads to
the activation of PKC and TGF- and, consequently, increased extracellular matrix (ECM)
synthesis. Abbreviation: GFAT, glutamine:fructose-6-phosphate-aminotransferase.
Figure 4.
Schematic drawing depicting the generation of advanced glycation end products (AGEs) and
downstream events. AGEs are formed by condensation of a sugar (R) such as glucose with a
reactive -amino (NH2) group of the protein; this process is followed by the formation of a
Schiff base, Amadori rearrangement, and a complex series of reactions. AGE:RAGE
(receptor for AGE) interactions lead to the generation of reactive oxygen species (ROS) and
the activation of protein kinase C (PKC), transforming growth factor (TGF-),
mitogenactivated protein kinase (MAPK), and transcription factors such as nuclear factor
B (NF-B), leading to increased synthesis of the extracellular matrix (ECM). Diabetic
injury is further amplified by the feedback loops of angiotensin II (Ang II) and cross-linking
of de novo synthesized excess ECM proteins with sugars. Abbreviations: AT1, Ang II
receptor; TGF-R, TGF- receptor.
Figure 5.
Sequence of events following AGE:RAGE (advanced glycation end products:receptor for
advanced glycation end products) interactions and excess glucose entry into the cell via
glucose transporter (GLUT). Diacylglycerol (DAG) and activated phospholipase C (PLC)
increase the expression and activity of protein kinase C (PKC), which modulates the
expression of a wide variety of genes that adversely affect glomerular pathophysiology,
thereby leading to increased vascular permeability, mesangial expansion, hyperfiltration,
and proteinuria. Abbreviations: Ang II, angiotensin II; e-NOS, endothelial nitric oxide
synthase; ET-1, endothelin 1; IP3, inositol trisphosphate; PAI-1, plasminogen activator
inhibitor 1; PIP2, phosphatidylinositol 4,5-bisphosphate; ROS, reactive oxygen species;

TGF-, transforming growth factor ; VEGF, vascular endothelial growth factor.
Figure 6.
Summary of events leading to the generation of cytosolic reactive oxygen species (ROS)
(R1) and mitochondrial ROS (R2). Extramitochondrial cytosolic ROS generation occurs
following increased glucose flux, activation of the polyol pathway, and AGE:RAGE
(advanced glycation end products:receptor for advanced glycation end products) interaction,
as well as via the NADPH oxidase system, such as NOX4. Mitochondrial matrix ROS and
reactive nitrogen species (RNS) are generated via the well-characterized electron-transport
chain, Complex IIV redox carriers, localized in the inner mitochondrial membrane. During
hyperglycemia, there is an increased donation of electrons (e) by powerful NADH- and
FADH2-reducing agents of Complex I and Complex II, respectively, characterized by
pumping of protons (H+) into the intermembrane space, which gives rise to an increased
mitochondrial membrane potential. As a result, the electron transport at complex III is
inhibited; the system backs up, and the half-life of the free-radical intermediates of
coenzyme Q (CoQ) is prolonged, which leads to an increase in the reduction of O2 to
superoxide (O2.) and in the production of ROS. The generated ROS and RNS release
cytochrome c, activate caspases, and induce apoptosis. They also modulate the activity of
angiotensin II (Ang II), protein kinase C (PKC), and transforming growth factor (TGF-),
which ultimately affect the synthesis of the extracellular membrane. Abbreviation: DAG,
diacylglycerol.
Figure 7.
Schematic depicting transforming growth factor (TGF-) and bone morphogenetic protein
7 (BMP-7) signaling. Activation of latent TGF- by glucose, advanced glycation end
products (AGEs), reactive oxygen species (ROS), and angiotensin II (Ang II) leads to the
generation of TGF-p, which binds first to type II and III serine/threonine kinase receptors
with recruitment and phosphorylation of type I. Activated heteromeric complex interacts
with Smad2/3 and co-Smad4. Smad2/3 are inhibited by Smad6/7, which are induced by
tumor necrosis factor . (TNF-) and interferon- (IFN-) signaling. The Smad2/3/4
complex translocates into the nucleus to initiate transcription of various extracellular matrix
(ECM) genes and connective tissue growth factor (CTGF). BMP-7, however, activates
Smad1/5/8, which bind to co-Smad4 and, upon translocation into the nucleus, induce Id
proteins that inhibit differentiation and DNA binding of some of the transcription factors,
thereby opposing the action of TGF-. Abbreviations: JAK, Janus kinase; MMP, matrix
metalloproteinase; NF-B; nuclear factor B; PKC, protein kinase C; STAT, signal

transducer and activator of transcription; TIMP, tissue inhibitor of metalloproteinase.
Figure 8.
Schematics depicting the sequence of events, following the interactions between advanced
glycation end products (AGEs) and angiotensin II (Ang II) with their respective receptors,
that lead to the generation of reactive oxygen species (ROS) and the activation of protein
kinase C (PKC) and Rap1 and Rho GTPases. Both Rap1 and Rho, in association with
guanine exchange factors (GEFs), are activated (GTP bound), whereas GTPase-activating
proteins (GAPs) hydrolyze Rap1 and Rho into the inactive (GDP-bound) state. Thus, these
small G proteins cycle between functional and nonfunctional states. Activated Rho GTP
leads to the induction of Rho kinase and the phosphorylation of Janus kinase/stress-activated
protein kinase (JNK/SAPK) kinase and phosphatase 1 (PPtase1), which then translocate into
the nucleus to initiate transcription via various transcription factors. Similarly, Rap1 GTP, in
association with another kinase known as B-Raf, causes the induction and phosphorylation
of extracellular signalrelated kinase (ERK) and mitogen-activated protein kinase/ERK
(MEK). Upon translocation into the nucleus the transcription of target genes is initiated.
Interestingly, the Rap1-ARAP1 (angiotensin II type 1 receptorassociated protein) complex
inhibits the activity of Rho kinase. Abbreviations: AP-1, activator protein 1; ATF, activating
transcription factor; CBP, cAMP response elementbinding (CREB) protein; ELK1, ets-like
gene 1; NF-B, nuclear factor B.
Figure 9.
Schematic drawing depicting local glomerular activation of the renin-angiotensin system
(RAS) by glucose flux and advanced glycation end products (AGEs). Both podocytes (Po)
and mesangial cells (Me) of the glomerulus express angiotensin II (Ang II) receptors (AT1).
Angiotensin I (AI) is synthesized from angiotensinogen (AGT) by the action of renin. AI is
converted into angiotensin II (Ang II) by angiotensin-converting enzyme (ACE). Ang II,
upon binding to its AT1 receptor, induces various nonhemodynamic effects, including
increased activity of transforming growth factor (TGF-) and expression of monocyte
chemotactic protein 1 (MCP-1), vascular endothelial growth factor (VEGF), and reactive
oxygen species (ROS). Abbreviations: Cap, capillary; En, endothelium; IL-6, interleukin-6;
TGF-R, TGF- receptor.
Figure 10.
Schematics depicting systemic activation of the renin-angiotensin system by glucose flux
and advanced glycation end products (AGEs). High-glucose flux and AGEs may induce
hypertrophy and hyperplasia of the juxtaglomerular apparatus (JGA), leading to increased

secretion of renin from stored cellular granules. This process causes the generation of a
potent vasoactive peptide known as angiotensin II (Ang II), which modulates the secretion
of adrenal aldosterone with Na+ retention, and it also induces constriction of the efferent
arteriole (EA). As a result of this mechanical stretch, systemic blood pressure rises, leading
to worsening of the hyperglycemic injury in various target organs or cells. Abbreviations:
AA, afferent arteriole; Cap, capillary; ACE, angiotensin-converting enzyme; AI, angiotensin
I; ATG, angiotensinogen.

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Annu Rev Pathol. 2011 ; 6: 395423. doi:10.1146/annurev.pathol.4.110807.092150.

A Glimpse of Various Pathogenetic Mechanisms of Diabetic

Copyright 2011 by Annual Reviews. All rights reserved

glomerular capillaries, which clinically manifest as hyperfiltration and microalbuminuria (3,

Well-described characteristic glomerular changes include mesangial cell proliferation and

In this review, we discuss pathogenetic mechanisms relevant to hyperglycemia-induced

CELLULAR UPTAKE OF GLUCOSE AND ITS PROCESSING INTO VARIOUS

conversion to glucose 6-phosphate (G6-P), which isomerizes to fructose 6-phosphate (F6-P);

glucuronic acid by myo-inositol oxygenase (MIOX), which is expressed exclusively in the

HYPERGLYCEMIA-INDUCED FORMATION OF ADVANCED GLYCATION

HYPERGLYCEMIA-INDUCED ACTIVATION OF PROTEIN KINASE C AND

PKC activation can influence a number of downstream pathophysiological changes, such as

REACTIVE OXYGEN SPECIES MODULATION OF OTHER

HYPERGLYCEMIA-INDUCED CELLULAR SIGNALING EVENTS

ROS are generated as a by-product of oxidative phosphorylation, in which electron donors

The relevance of extramitochondrial ROS to the pathogenesis of diabetic nephropathy is

supported by studies in which overexpression of cytoplasmic CuZnSOD reduced glomerular

Similar to the mitochondrial electrontransport system, the NADPH-oxidase system can be

TRANSFORMING GROWTH FACTOR CELLULAR SIGNALING IS CRITICAL

FIBROSIS IN DIABETIC NEPHROPATHY

TGF-1 signaling is initiated by numerous mediators generated under high-glucose

of diabetes (86, 87). That administration of neutralizing anti-TGF-1 antibodies prevents

Bone morphogenetic protein 7 (BMP-7), a member of the TGF- superfamily, acts in

EMERGING RELEVANCE OF GTP-BINDING PROTEINS AND CELL-CYCLE

intermediary between the activated/phosphorylated growth factor receptor and MEK,

ROLE OF INTRAGLOMERULAR HYPERTENSION IN THE AMPLIFICATION

OF HYPERGLYCEMIA-INDUCED RENAL INJURY IN DIABETIC

response occurs upon reduction of the intraglomerular hypertension in states of

nephropathy also underscores the importance of ACE2 and intraglomerular hemodynamics

PATHOGENETIC MECHANISMS RELEVANT TO TUBULOINTERSTITIAL

Some mechanisms relevant to tubulointerstitial pathobiology may be similar to those

mechanisms (147). First, a glomerular filtrate containing excessive amounts of profibrogenic

Another recently identified mechanism by which tubulointerstitial damage can occur in

molecular pathology. Eur. J. Clin. Investig. 2004; 34:785796. [PubMed: 15606719]

D-chiroinositol. Biochem. J. 2001; 300:313320. [PubMed: 11716759]

diabetic nephropathy. J. Am. Soc. Nephrol. 2003; 14:S221S226. [PubMed: 12874435]

1997; 7:7579. [PubMed: 9024640]

ameliorates progression of diabetic nephropathy. J. Am. Soc. Nephrol. 2003; 14:699703.

factor in mouse podocytes. Diabetes. 2004; 53:29392349. [PubMed: 15504975]

tubulointerstitial disease in diabetic nephropathy. (a) A normal glomerulus. (b) Thickened

kinase; MCP-1, monocyte chemoattractant protein 1; NF-B; nuclear factor B; RNS,

inhibitor 1; PIP2, phosphatidylinositol 4,5-bisphosphate; ROS, reactive oxygen species;

metalloproteinase; NF-B; nuclear factor B; PKC, protein kinase C; STAT, signal

hypertrophy and hyperplasia of the juxtaglomerular apparatus (JGA), leading to increased

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